Project Summary Rabs constitute the largest branch of the Ras GTPase superfamily, with ten members in yeast and more then 60 in mammalian cells. They serve as key nodes in the regulation of membrane traffic, each typically controlling several different aspects of a specific stage of membrane traffic by recruiting diverse effector proteins such as cytoskeletal motors, vesicle tethering proteins and regulators of SNARE complex assembly. Rabs are activated by specific guanine nucleotide exchange factors (GEFs) that catalyze the displacement of GDP and binding of GTP and are inactivated by GTPase activating proteins (GAPs) that stimulate the slow intrinsic rate of GTP hydrolysis. We have proposed that adjacent Rabs on a traffic pathway are networked to one another through their regulators. The postulated effect of these counter-current cascades is a programmed series of abrupt Rab conversions that lead to changes in the functional identity of the membrane as it flows along a pathway. Rabs are also thought to define the anterograde versus retrograde directionality of vesicular transport. In addition, Rab GEF-effector interactions are believed to generate positive feedback loops that promote vesicle maturation. While the roles of individual elements of the regulatory network have been examined, these proposals have not yet been tested at a more global, integrated level. We propose three specific aims that will address important aspects of the Rab regulatory network: 1. We will test the effects of rewiring a Rab regulatory circuit to evaluate its role in establishing organelle identity and pursue a novel inhibitory role for a coiled-coil vesicle tether that has emerged from these studies 2. We will directly test the role of Rabs in the control of the directionality of membrane traffic 3. We will determine the role of a Rab GEF-effector interaction in secretory vesicle maturation